1. Technical Field
The present invention is concerned with improved methods and apparatus relating to confocal scanning optical microscopy and, in particular, to a confocal scanning laser microscope having no moving parts and the capability of providing real time video images of the scanned object.
2. Discussion of the Prior Art
Scanning optical microscopes and, in particular, confocal scanning optical microscopes, are well known in the prior art. For example, a confocal scanning optical microscope is disclosed in U.S. Pat. No. 4,198,571 (Sheppard). An excellent treatise on scanning optical microscopes is found in the text by Tony Wilson entitled "Theory and Practice of Scanning Optical Microscopy", Academic Press, 1984. Chapter one of that text provides an informative background discussion on scanning optical microscopes, and confocal scanning optical microscopes in particular, and is expressly incorporated herein in its entirety by this reference. Typically, the confocal scanning laser microscope includes a stationary laser beam coinciding with the optical axes of two objective lenses, the first of which focuses the beam onto a diffraction-limited spot at the specimen, the second of which images the beam emerging from the other side of the specimen onto a pinhole in front of a photodetector. In order to produce an image of the specimen, the photodetector modulates the intensity of a television raster scan performed in synchronization with a geometrically similar mechanical scan of the specimen through the laser beam. The term "confocal" is used to indicate that both objective lenses are focused on the same point on the specimen. The pinhole acts to reduce the depth of focus of the instrument while conferring an increase in lateral resolution of 1.4 times as compared with a conventional imaging microscope. Not only is the depth of field reduced, bu the pinhole rejects light reflected by de-focussed planes; that is, the pinhole greatly attenuates light originating from planes displaced from the plane of focus, or from adjacent regions within the plane of focus (i.e., due to scattering). In his text, Wilson considers the possibility of scanning the laser beam as an alternative to mechanically scanning the object and concludes that the latter is preferable. Although recognizing that beam scanning can be rapid and, therefore, many whole-picture images can be formed per second, Wilson concludes that mechanical object scanning produces higher quality images with less distortion. However, it should be noted that high quality microscope objective lenses are readily available, and that these do not significantly compromise resolution over the field. It should also be noted that in low-level fluoroscopy, where light is limited by fluorochrome bleaching, there is no microscope that provides ultimate resolution because of the mandatory signal to noise tradeoff between spatial and temporal resolution. To obtain good spatial resolution it is necessary to collect photons over a long time interval. Conversely, in order to obtain good temporal resolution it is necessary to collect photons over a large area. In other words, comparative resolution is a moot issue in choosing between beam scanning and mechanical scanning of the specimen. Wilson points out other presumed advantages of mechanical scanning of the object or specimen, such as simplified lens design when the optical path is stationary, easily achieved interference scanning microscopy without the necessity of matched optics, and less complexity than is associated with beam deflection. I have found that not all of these so-called advantages of mechanical object scanning are in fact true advantages. Rather, the main disadvantage of beam scanning relates specifically to the nature of confocal scanning microscopes and concerns a suitable arrangement for scanning the pinhole so that it remains conjugate with the laser spot as that spot sweeps a raster pattern in the specimen at video scan rates.
A condensed quantitative treatment of confocal microscope imaging is presented in another article by Wilson entitled "Imaging Properties And Application Of Scanning Optical Microscopes", appearing in "Applied Physics, Vol. 22, pages 119-128 (1980), particularly, at pages 124 through 127". In this article Wilson points out that the image of a hypothetical point object is sharper for confocal scanning microscopes (e.g., by as much as forty percent at the fifty percent intensity point) than for a conventional microscope. The mathematical expression for the point spread function intensity for the confocal case is approximately the square of the expression for intensity for the conventional microscope, resulting in a sharper point. It is also pointed out by Wilson that the confocal microscope has less attenuation of the higher spatial frequencies than does a conventional microscope. In the Wilson text "Theory And Practice Of Scanning Optical Microscopy" referred to above, Wilson discusses the optical sectioning properties of the two types of microscopes. The important point here is that a conventional light microscope provides optical sectioning by virtue of its finite depth of focus, but all of the out-of-focus light still participates in the final image; whereas the confocal scanning microscope provides optical sectioning because it sharply attenuates the out-of-focus light, and only in-focus light contributes to the image.
The optical sectioning capability is considered to be particularly important for live tissue fluorescent microscopy where stray out-of-plane fluorescence greatly impairs image quality.
Another type of confocal scanning microscope providing optical sectioning is generally referred to as the Tandem Scanning Reflected Light Microscope (TSRLM) and is described in an article appearing in Science Magazine, Vol. 230, Dec. 13, 1985 at pages 1258, 1259 and 1262. In the TSRLM an incident light beam is focused through an input group of holes in a rotating disk and passes through an objective lens which focuses the beam within the specimen. Light reflected from the focus plane passes back through the objective, is focused on a set of precisely positioned output holes in the disk which are optically congruent with the input holes via the rest of the system, and passes through to an eyepiece. Light from planes other than the focus plane does not get through the output holes and is blocked by the rotating disk. This system tends to be quite complex.
In terms of optical performance there are definite advantages to the two types of confocal scanning microscopes described above as compared to non-confocal microscopes. Their somewhat limited use to date appears to be due to a number of problems. Specifically, existing laser confocal scanning microscopes mechanically scan the specimen in a raster pattern and are inherently mechanically complex devices which are difficult to maintain and use. In addition, mechanical specimen scanning is inherently a relatively slow process so that image acquisition requires something on the order of ten seconds. Speeds required to produce high resolution images in real time would require unacceptable accelerations for live specimens. Although the Tandem Scanning Reflected Light Microscope is a real time device, it is very complex and even more difficult to maintain, and tends to be more impractical than existing laser confocal scanning microscopes.
Additional background in the art of scanning light microscopy may be found in the following publications: Brakenhoff et al, "Developments In High Resolution Confocal Scanning Light Microscopy (CSLM)", appearing in "Scanned Image Microscopy" edited by E. A. Ash, Academic Press, 1980; Valkenburg et al, "Confocal Scanning Light Microscopy Of The Escherichia Coli Nucleoid: Comparison With Phase-Contrast And Electron Microscope Images", appearing in the Journal of Bacteriology, February 1985, Vol. 161, No. 2, pages 478-483; Brakenhoff et al, "High-Resolution Confocal Scanning Light Microscopy In Biology", appearing in Analytical Chimica Acta, Vol. 163 (1984), pages 231-236; Brakenhoff et al, "Three-Dimensional Chromatin Distribution In Neuroblastoma Nuclei Shown By Confocal Scanning Laser Microscopy", appearing in Nature, Vol. 317, October 24, 1985, pages 748, 749.
From the foregoing it will be appreciated that the prior art confocal scanning laser microscope obtains its improved imaging properties by utilizing a pinhole to admit light which is conjugate to the diffraction-limited laser illuminated spot in the object while simulatenously rejecting light from the other object regions. To map out an image of an extended object, the image is mechanically scanned through the beam in a raster pattern. The resulting instrument is mechanically complex and relatively slow. The Tandem Scanning Microscope operates in real time but involves even greater mechanical complexity. It would appear, therefore, to be quite useful to have a microscope that operates in real time (like the Tandem Scanning unit), which has the sequential scanning type operation of a confocal laser scanning device, and which preserves the improved imaging characteristics of both but does not have the mechanical complexity of either. In my efforts to provide just such a microscope, my first efforts were directed toward speeding up the confocal laser scanning microscope. This may be done by replacing mechanical specimen scanning with some type of laser beam steering controlled to sweep the desired video raster pattern along the specimen. In addition to a variety of mechanical scanners utilizing rotating mirror-faceted wheels and electromechanically activated assemblies (e.g., galvanometer-type devices), it is known to utilize acousto-optic effects to steer a laser beam. Acousto-optic deflectors are employed in laser printers, as well as other devices, and utilize a piezoelectric transducer excited at approximately 50 MHz to establish a pressure wave in a transparent crystal which acts as a diffraction grating because the index of refraction changes with pressure. The laser beam is incident on the crystal at approximately right angles to the pressure wave. Typically, approximately seventy percent of the laser beam is diffracted into a first order beam having an exit angle from the crystal which is proportional to the inverse of pressure wavelength. By varying the piezoelectric excitation frequency with a voltage controlled oscillator, or other means, the beam angle can be varied arbitrarily (e.g., randomly or in a scanning mode). Two of these acousto-optic deflectors positioned in tandem at right angles are capable of sweeping the beam in a two-dimensional raster pattern at television scan rates. Resolution and repeatability of the beam angle can be on the order of one part in two thousand. Scan patterns having five hundred horizontal lines with five hundred points per line are typical. Output beam maximum deflection angle on the order of two degrees could be increased with lenses, if necessary, before filling the objective lens rear entrance pupil and focusing onto a microscopic spot which illuminates the object. A beam scanning arrangement of this type is described by Suzuki et al, "Development Of A Real Time Scanning Laser Microscope For Biological Use", appearing in Applied Optics, Vol. 25, No. 22, Nov. 15 1986, pages 4115 through 4121. Clearly, then, the problem of rapidly and controllably scanning a laser beam across an object in a microscope has been substantially solved. However, in order to retain confocality, there still remains the problem of simultaneously scanning the pinhole so that it remains conjugate with the laser spot being swept in a raster pattern across the specimen at video rates. It is with this problem that the present invention is concerned. It is of more than passing interest to note that Suzuki et al consider the possibility of confocality for their scanning microscope; however, they decide against it because prior art confocal systems cannot be satisfactorily operated to provide a real-time, continuously moving image.